2′-N-(Pyren-1-yl)acetyl-2′-Amino-α-L-LNA: Synthesis and Detection of Single Nucleotide Mismatches in DNA and RNA Targets

ChemBioChem ◽  
2007 ◽  
Vol 8 (10) ◽  
pp. 1122-1125 ◽  
Author(s):  
T. Santhosh Kumar ◽  
Jesper Wengel ◽  
Patrick J. Hrdlicka
Keyword(s):  
Single Nucleotide ◽  
Dna And Rna ◽  
Rna Targets

Highly sensitive detection of rare mutant alleles by combining argonaute-based enrichment and XNA-PCR.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14605-e14605
Author(s):  
Jinzhao Song ◽  
Michael Joseph Powell ◽  
Wei Liu ◽  
Junman Chen ◽  
Haim Bau
Keyword(s):  
Nucleic Acids ◽  
High Efficiency ◽  
Elevated Temperatures ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Detection Methods ◽  
Single Nucleotide ◽  
Complementary Dna ◽  
Dna And Rna ◽  
Rna Targets

e14605 Background: Characterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early disease detection, genotyping, and personalized therapy. Detection of alleles of clinical interest is often challenged by their low concentration and sequence homology with the much more abundant wildtype nucleic acids. Methods: Argonuate (Ago) from the thermophilic bacterium Thermus thermophilus ( TtAgo) utilizes short DNA guides to specifically cleave complementary DNA and RNA targets. We found that under optimized conditions, TtAgo cleaves DNA and RNA complementary to the guide DNA with high efficiency, but spares nucleic acids with a single nucleotide mismatch at and around its catalytic site with high sensitivity. Based on these findings, we designed a new multiplexed enrichment assay, dubbed NAVIGATER (Nucleic Acid enrichment Via DNA Guided Argonaute from Thermus thermophilus), that utilizes TtAgo, to specifically cleave perfectly complementary DNA and RNA while sparing alleles of interest. Results: NAVIGATER greatly increases the fractions of rare mutant alleles with single nucleotide precision enhancing the sensitivity of downstream detection methods such as XNA-PCR. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and over ten-fold improved sensitivity of XNA-PCR, enabling multiplex detection of KRAS and EGFR mutants at 0.01% fractions. Conclusions: NAVIGATER has important advantages over other mutant allele enrichment assays such as the ones based on CRISPR-Cas. It does not require the target to contain a protospacer-adjacent motif; is a true (turnover) catalyst; can cleave both DNA and associated exosomal RNA targets, improving sensitivity; and can operate at elevated temperatures for higher selectivity and compatibility with detection schemes.

scholarly journals Asymmetric Divergence in Transmitted SNPs of DNA Replication/Transcription and Their Impact on Gene Expression in Polyploid Brassica napus

2021 ◽  
Vol 12 ◽  
Author(s):  
Minqiang Tang ◽  
Juanling Li ◽  
Xu Hu ◽  
Lu Sun ◽  
MMU Helal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Brassica Napus ◽  
Genome Evolution ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Expression Levels ◽  
Dna And Rna ◽  
Polyploid Genome ◽  
Gene Expression Levels

The marked increase in plant genomic data has provided valuable resources for investigating the dynamic evolution of duplicate genes in polyploidy. Brassica napus is an ideal model species for investigating polyploid genome evolution. The present study comprehensively analyzed DNA and RNA variation of two representative B. napus inbredlines, Zhongshuang11 and Zhongyou821, and we investigated gene expression levels of An and Cn subgenomes in multiple tissues of the two lines. The distribution of transmitted single nucleotide polymorphisms (SNPs) was significantly different in two subgenomes of B. napus. Gene expression levels were significantly negatively correlated with number of variations in replication and transcription of the corresponding genes, but were positively correlated with the ratios of transmitted SNPs from DNA to RNA. We found a higher density of SNP variation in An than that in Cn during DNA replication and more SNPs were transmitted to RNA during transcription, which may contribute to An expression dominance. These activities resulted in asymmetrical gene expression in polyploid B. napus. The SNPs transmitted from DNA to RNA could be an important complement feature in comparative genomics, and they may play important roles in asymmetrical genome evolution in polyploidy.

scholarly journals Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression

2010 ◽  
Vol 56 (12) ◽  
pp. 1845-1853 ◽  
Author(s):  
Guido Hennig ◽  
Mathias Gehrmann ◽  
Udo Stropp ◽  
Hiltrud Brauch ◽  
Peter Fritz ◽  
...  
Keyword(s):  
Extraction Method ◽  
Nucleotide Polymorphisms ◽  
Automated Extraction ◽  
Single Nucleotide ◽  
Normal Tissues ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna And Dna ◽  
Formalin Fixed

BACKGROUND There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1–25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%–100% of tumor tissues and 21%–100% of normal tissues. The 7 SNPs were successfully genotyped in 91%–97% of tumor and 94%–97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%–99.5%. CONCLUSIONS This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

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